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The Interaction of Ultraviolet-B Radiation and Water Deficit in Two Arabidopsis thaliana Genotypes
Annals of Botany 85: 571±575, 2000 doi:10.1006/anbo.1999.1085, available online at http://www.idealibrary.com on
S H O R T CO M M U N I CAT I O N
The Interaction of Ultraviolet-B Radiation and Water De®cit in Two Arabidopsis thaliana Genotypes A N N A - M A RY S C H M I D T {, D O U G L A S P. O R M RO D{* , N I G E L J. L I V I N G S TO N { and S A N TO S H M I S R A { {Department of Biochemistry and Microbiology, Centre for Forest Biology, University of Victoria, P.O. Box 3020, Victoria, B.C., Canada, V8W 3N5 and {Department of Biology, Centre for Forest Biology, University of Victoria, P.O. Box 3020, Victoria, B.C., Canada, V8W 3N5 Received: 18 May 1999 Returned for revision: 28 September 1999 Accepted: 8 December 1999 It has been demonstrated, in both herbaceous and woody species, that tissue hydration resulting from exposure to drought is less pronounced if plants are concurrently exposed to ultraviolet-B radiation (UV-B). An explanation for the mechanisms underlying this phenomenon has been elusive. Arabidopsis thaliana (L.) Heynh. genotypes, defective in speci®c defences against UV-B exposure, may permit more insightful study of drought-UV-B interactions than is possible with genetically uniform plants. Arabidopsis has a rosette stature and has predominantly abaxial stomata. Thus, it is dicult to investigate its stomatal behaviour and gas exchange using conventional techniques and instrumentation. In this study, the relative abundance of 13C and 12C in leaf tissue (d 13C) was used as a means of determining water use eciency (WUE) and the relative balance, at the site of carbon ®xation, between CO2 supply and demand. UV-B insensitive (Ler) and sensitive ( fah1) Arabidopsis genotypes were raised in a growth chamber and exposed to 6 kJ m ÿ2 d ÿ1 UV-B irradiation and subjected to drought. In both genotypes, leaf desiccation was less pronounced than that of control plants that were subjected to drought but not exposed to UV-B. The relatively low (more negative) leaf d 13C values (indicating low WUE), but high dry matter production of the UV-B exposed plants suggest that their higher leaf water content was not primarily due to stomatal closure. We propose that the mechanisms underlying the maintenance of higher leaf water content involved UV-B and water stress induced biosynthesis of # 2000 Annals of Botany Company stress proteins and compatible osmolytes. Key words: Arabidopsis thaliana, ultraviolet-B, water de®cit, stable carbon isotopes, d dehydration, dehydrin.
I N T RO D U C T I O N All components of the biosphere are exposed to ultravioletB radiation (UV-B) at intensities that vary with the solar angle and the thickness of the stratospheric ozone layer (Jansen, Gaba and Greenberg, 1998). Research on the adaptation of plants to UV-B has revealed responses that suggest that UV-B is involved in the induction of multistress defences. Evidence of interactions between UV-B exposure and water stress in plants has emerged in recent years, but the mechanisms involved have received little attention. In ®eld-grown soybean, decreases in productivity following UV-B exposure were moderated by soil water de®cits (Sullivan and Teramura, 1990). The soybean cultivar most yield-sensitive to UV-B when well watered was least sensitive to UV-B when subjected to water stress. In contrast, a UV-B tolerant cultivar was sensitive to UV-B when also subjected to water stress (Teramura, Sullivan and Lydon, 1990). The interaction of soil water de®cit and UV-B stresses in cowpeas resulted in bene®ts from the combined stresses in terms of greater growth and * For correspondence. Fax (250) 721-7120, e-mail [email protected]
0305-7364/00/040571+05 $35.00/00
13
C, stomatal opening, tissue
development compared with exposure to single stresses (Balakumar, Hani Babu Vincent and Paliwal, 1993). Increased exposure to UV-B alleviated drought stress in Mediterranean pines (Petropoulou et al., 1995; Manetas et al., 1997). However, exposure to both UV-B and water stress led to decreased growth in cucumber and radish but protein content was increased by the combined stresses (Tevini, Iwanzik and Teramura, 1983). These initial reports indicated that both genotypic dierences and assimilate utilization are involved in the interaction of UV-B and water stress. The objective of this study was to investigate the interactive eects of exposure to drought and UV-B radiation on the growth and water relations of Arabidopsis. Speci®cally, we sought to determine whether exposure to UV-B limits tissue dehydration when plants are also subjected to drought stress. We hypothesized that such a response might come about because UV-B exposure stimulates the production of compatible osmolytes and stress proteins, thus allowing plants to maintain tissue turgor without closing their stomata. We speculated that this response would be more pronounced in a UV-B sensitive genotype. # 2000 Annals of Botany Company
572
Schmidt et al.ÐUV-B-Water Stress Interactions in Arabidopsis
Arabidopsis thaliana mutants defective in the synthesis of protective UV-B absorbing compounds have been widely used to study injury and defences against UV-B (Bharti and Khurana, 1997). The limited epidermal attenuation of UV-B radiation in these mutants, relative to that of wildtype genotypes, may help elucidate the mechanisms involved in UV-B interactions with other stresses in the underlying mesophyll tissue (Ormrod, Landry and Conklin, 1995). Our study utilized the fah1 mutant which, in contrast to the wild-type ecotype Landsberg erecta, lacks the ability to synthesize UV-absorbing sinapate esters and is thus highly sensitive to UV-B exposure (Landry, Chappel and Last, 1995). Because conventional gas exchange measurements are dicult to carry out on small plants such as Arabidopsis, we used measurements of tissue d 13C as an alternative. The relative abundance of 12C to 13C (d 13C) provides an integrated measure of Ci (the internal CO2 concentration) over the period during which CO2 is assimilated by the plant (Farquhar et al., 1982). Values of Ci vary among plants because of variation in stomatal conductance to CO2 and variation in photosynthetic capacity (chloroplast demand for CO2). Assuming that the concentration and isotopic composition of ambient CO2 is stable over the period of interest, the isotopic composition of leaf tissue should, therefore, re¯ect the balance between CO2 supply (and water lost) and assimilation. Discrimination against 13C typically decreases in response to the progressive depletion of rooting medium water that leads to stomatal closure and decreased Ci (Knight, Livingston and Van Kessel, 1994; Livingston et al., 1999). This is re¯ected in higher d 13C values and greater water use eciency (WUE). M AT E R I A L S A N D M E T H O D S Two independent experiments were performed, each using all combinations of two genotypes, two UV-B dosages, and two watering regimes. In each experiment, each of the eight treatment combinations had two replicate 12-cm diameter pots of each genotype of Arabidopsis thaliana (L.) Heynh. Ler and fah1 seedlings were propagated in a controlled environment chamber at 258C under cool white ¯uorescent lamps with a continuous photosynthetic photon ¯ux density of 150 mmol m ÿ2 s ÿ1. Weighed samples of seeds (1 mg 50 seeds) were suspended in tubes containing 0.1% (w/v) agar in water to facilitate uniform distribution of approx. 50 seeds on the surface of a wetted ®ne-textured synthetic rooting medium. Seeded pots were covered with transparent ®lm for 7 d. Seedlings were grown for a further 6 d after the ®lm was removed and then exposed for 10 d to 6 h d ÿ1 of either 0 UV-B (Mylar ®lm between lamps and plants) or 6 kJ m ÿ2 s ÿ1 (cellulose acetate ®lm between lamps and plants) biologically eective UV-B (Caldwell and Flint, 1997) from UV-B-emitting ¯uorescent lamps (UV-B 313, Q-Panel, Cleveland, OH, USA) added to the cool white ¯uorescent lamps. Complete nutrient solution was added to all pots from 11 to 13 d after seeding. Two rooting medium watering regimes were administered. Half the pots were wetted to ®eld capacity every second day; water was withheld from the other pots 14 d
after seeding. This discontinuation of watering, calibrated for the pot size, rooting medium and plant numbers, resulted in the onset of measurable leaf dehydration in control plants by the sixth day (19 d after seeding) of the UV-B exposure period. Seedlings were thinned to 30 per pot just before dierential treatments were initiated. The shoots of ®ve randomly selected seedlings were removed at each of six harvests, starting at the end of the daily UV-B exposure 14 d from seeding ( pretreatment) and continuing every second day to 24 d from seeding (10 d of treatment). Fresh shoots were weighed immediately, dried at 708C for 48 h, and reweighed to determine shoot water content at harvest. Dried shoot samples from the ®rst experiment were pulverized and subsamples analysed for d 13C by continuous ¯ow isotope ratio mass spectroscopy on a TracerMass mass spectrometer interfaced with a RoboPrep sample preparation unit (Europa Scienti®c, Crewe, UK). A peptide with the sequence CTGEKKGIMDKIKEKLPGQH was synthesized at the University of Victoria Protein Micro-Chemistry Centre, Victoria, Canada. This synthetic peptide containing the dehydrin carboxy terminus consensus sequence KIKEKLPG (Close, Raymond and Moonan, 1993) was used to produce antibodies. The peptide was coupled to maleimide-activated KLH carrier protein using the Imject1 kit according to the manufacturer's directions. The peptide-KLH conjugate was mixed with Freunds adjuvant and injected into rabbits; antipeptide antibody titres and speci®city were determined by ELISA with the free dehydrin peptide. Protein extraction and immunoblot analysis utilized the same growing conditions as above. Shoots for protein analysis were harvested 7 d after the beginning of UV-B treatment and immediately frozen in liquid nitrogen and stored at ÿ808C. There were control and UV-B treated plants only, and no water stressed plants. Total heat-stable protein extracts were prepared by a modi®cation of the method of Close et al. (1993). Tissue samples stored at ÿ808C were ground to a ®ne powder in liquid nitrogen with a mortar and pestle. Extraction buer (30 mM TES, pH 7.5; 50 mM NaCl; 0.5 mM PMSF) was added to ground tissue in a microfuge tube, vortexed and centrifuged at 13 800 g for 20 min. The supernatant was incubated at 908C for 30 min, transferred to ice, then centrifuged at 9000 g for 10 min at 48C. Heat stable proteins contained within the supernatant were quanti®ed by Bio-Rad protein assay. Duplicate SDS-PAGE gels were prepared and run according to Sambrook, Fetsch and Maniatis (1989). One gel was transferred to a nitrocellulose membrane using a Bio-Rad MiniProtein II Wet Blotting system, blocked with 3% (w/v) skim milk in TBS buer and incubated with rabbit anti-dehydrin polyclonal antibodies. Membranes were then incubated with goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma). Secondary antibody was detected using 4-nitroblue-tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP). Duplicate gels were Coomassie stained. Immunoblot analysis using pre-immune serum was performed, and no proteins were detected.
Schmidt et al.ÐUV-B-Water Stress Interactions in Arabidopsis R E S U LT S A N D D I S C U S S I O N Well watered Arabidopsis plants had growth responses to UV-B as would be expected for these genotypes (Landry et al., 1995). The shoot dry weight of Ler was unaected by UV-B while that of fah1 was decreased by 25±30% (Table 1). The shoot water content at harvest was uniformly near 90% in all well-watered treatments. In contrast, in plants subjected to drought, there were signi®cant declines in both water content and dry matter and clear dierences between UV-B treatments. These became most pronounced after 8 d of drought treatment. Six days after drought was imposed, large dierences in water content were not seen between genotypes across the UV-B treatments, but, within a given genotype, those plants not exposed to UV-B had the lowest water contents (Table 1). Dierences in dry matter production of droughtstressed plants across UV-B treatments were not signi®cant within genotypes. After 8 d, the shoot water content of drought-stressed fah1 plants exposed to UV-B was almost six-times as high as those that were not exposed. Despite these dierences in water content, there was no signi®cant dierence in dry matter production. Shoot water contents of Ler plants were lower than those measured after 6 d, but there was not a signi®cant dierence in water content between UV-B treatments. The d 13C data indicate that UV-B exposure, water de®cits and genotype had individual and interactive eects on carbon isotope discrimination (Fig. 1). In both genotypes, UV-B exposure resulted in greater discrimination against 13C (i.e. more negative d 13C values) in both well watered and drought-stressed plants. Dierences in sensitivity to UV-B exposure between genotypes became more pronounced over time. The lower d 13C values in fah1 plants, when exposed to UV-B, were consistent with their lack of protective UV-B absorbing compounds, and their lower productivity than Ler plants under either well watered or water stressed conditions. In both genotypes, the d 13C values for plants not exposed to UV-B but subjected to drought probably re¯ected stomatal closure
573
and hence a reduction in Ci. In both genotypes, dierences in d 13C between well watered and water stressed plants were less pronounced when plants were exposed to UV-B. This suggests that UV-B exposure might have oset some of the eects of water stress or vice versa. The low d 13C values in droughted fah1 plants exposed to UV-B suggests that the Ci in these plants was relatively high. There are alternative explanations. UV-B exposure might not have induced stomatal closure and therefore the supply of CO2 to the site of carboxylation was maintained. In a previous study we obtained results that suggested that exposure to UV-B did not induce stomatal closure in well-watered Ler or fah1 Arabidopsis (Ormrod, Schmidt and Livingston, 1997). If this was the case, the data suggest that UV-B exposure must have been associated with the production of osmolytes which allowed these plants (in marked contrast to those not exposed to UV-B) to maintain high water contents even when subjected to severe drought (Table 1). An alternative explanation is that exposure to UV-B might have suciently damaged the plant's photosynthetic machinery, leading to decreased demand for CO2 and hence high Ci. This latter explanation is unlikely because UV-B exposure did not bring about a signi®cant reduction in dry matter production in droughted plants (Table 1). Immunoblot analysis revealed that a protein (approx. 60 kDa), recognized by antibodies directed against a synthetic peptide containing the dehydrin consensus sequence, was induced by UV-B in both Ler and fah1 genotypes of Arabidopsis; levels of the protein increased nominally in UV-B treated Ler plants compared to the controls, whereas the increase in UV-B treated fah1 plants was far more substantial (Fig. 2). The accumulation of dehydrins in response to UV-B radiation may occur more rapidly than would a response to slow dehydration by water stress, contributing to the less pronounced water stress-induced tissue dehydration by concurrent exposure to UV-B radiation. As well, it has been proposed that dehydrins may act synergistically with compatible solutes to stabilize macromolecules, thereby stabilizing the protoplasm (Close, 1996).
T A B L E 1. Shoot dry weight and water content at harvest in well watered or drought stressed A. thaliana genotypes Ler and fah1 subjected to 0 or 6 kJ m ÿ2 d ÿ1 UV-B irradiation Well watered
Drought stressed
UV-B dose (kJ m ÿ2 d ÿ1)
Dry weight (mg per plant)
Water content (%)
6d Ler Ler fah1 fah1
0 6 0 6
5.54 + 0.22a 5.37 + 0.20a 5.32 + 0.50a 3.94 + 0.31b
90.4 + 0.2a 90.3 + 0.5a 90.5 + 0.2a 90.2 + 0.2a
4.42 + 0.55ab 4.15 + 0.53abc 3.09 + 0.37c 3.62 + 0.14bc
85.3 + 0.5b 87.9 + 0.6a 83.9 + 0.4b 88.1 + 1.0a
8d Ler Ler fah1 fah1
0 6 0 6
6.97 + 0.22b 6.36 + 0.33b 8.40 + 0.41a 5.80 + 0.45b
89.9 + 0.3a 89.6 + 1.0a 89.8 + 0.3a 89.3 + 0.0a
3.60 + 0.50b 5.04 + 0.49a 3.23 + 0.30b 3.40 + 0.23b
55.8 + 16.7a 69.2 + 9.4a 12.7 + 3.1b 75.2 + 6.4a
Duration Genotype
Dry weight (mg per plant)
Water content (%)
Means of two experiments + s.e. Mean separation within durations and watering regimes by paired comparison t-tests (P 0.05). Means followed by the same letter within 6 or 8 d and dry weight or water content are not signi®cantly dierent.
574
Schmidt et al.ÐUV-B-Water Stress Interactions in Arabidopsis −UV
6 days −32
+W
−W
−UV
+UV +W
−W
−32
−34 −35 −36
A Ler
−36
−UV +W
−W
+W
−W
−32
+W
−W
+UV +W
−W
−33 δ13C (%0)
δ13C (%0)
B fah 1
−UV
+UV
−33 −34 −35 −36
−34 −35
8 days −32
−W
+W
−33 δ13C (%0)
δ13C (%0)
−33
−W
+W
+UV
−34 −35
C Ler
−36
D fah 1
FIG. 1. Stable carbon isotope composition (d 13C) of shoots after 6 and 8 d of treatment in control (ÿUV) and UV-B irradiated (UV) Arabidopsis thaliana genotypes (A, C) Ler and (B, D) fah1. Plants were either watered to ®eld capacity every second day (w) or subject to drought stress (ÿw). Means and s.e.s of two replicate pots, ®ve shoots per pot, from the ®rst experiment.
Both Ler and fah1 genotypes, when subjected to drought but exposed to UV-B, maintained relatively high shoot water contents (Table 1), even though the d 13C data indicate that there was not substantial stomatal closure. Thus it is likely that leaf water content was maintained as a result of the induction by UV-B of stress-related proteins and osmotic adjustment. The production and accumulation by plants of low-molecular-weight solutes, called compatible osmolytes, is a widespread adaptive response to water stress. These organic compounds are osmotically active and include sugar alcohols, some amino acids and quaternary ammonium compounds (Taylor, 1996). They have important roles in osmotic adjustment, protection of macromolecules and metabolite storage during times of stress (Wanek and Richter, 1997). Most compatible osmolytes are also eective hydroxyl radical scavengers (Smirno and Cumbes, 1989) and help protect cells against the hydroxyl radicals generated by both water and UV-B stress. The accumulation of compatible osmolytes allows cell turgidity to be maintained in the face of water de®cits in the rooting medium. Aside from compatible solute accumulation, biochemical adaptations to stress also include changes in gene expression. Emerging evidence indicates that numerous environmental cues (dehydration, heat shock, low temperatures)
cause similar proteins to accumulate. Examples of such stress-related proteins include members of the dehydrin protein family and various families of heat shock proteins. Dehydrins are among the more commonly identi®ed proteins induced by water de®cits. In addition, a relationship between dehydrin accumulation and adaptation to stresses that have a dehydrative component such as freezing and cold stress has been reported (Arora and Wisniewski, 1994; Close, 1996, 1997). Our ®nding in this and the previous study (Ormrod et al., 1997), that UV-B exposure does not appear to induce pronounced stomatal closure in Arabidopsis, implicates a potential dehydrative eect in UV-B response. The induction of proteins similar to heat shock proteins has been reported in Vigna sinensis seedlings exposed to UV-B (Nedunchezhian, Annamalainathan and Kulandaivelu, 1992). Large increases in the levels of three groups of proteins in the range of 70, 53 and 16 kDa, similar to those observed after heat shock treatment, were observed. UV-B radiation may provide a certain amount of cross-adaptation to other environmental stresses and, like other stresses, may induce sets of proteins having a protective role. Our results strongly suggest that UV-B exposure moderates the eects of water stress and demonstrate the need for further research to establish the relations between plant
Schmidt et al.ÐUV-B-Water Stress Interactions in Arabidopsis LC
LU
FC
FU
kDa 62 > 47.5 >
32.5 > FIG. 2. Immunoblot of dehydrin proteins present in Arabidopsis genotypes Ler and fah1; 5 mg total protein loaded per lane. LC (Ler control); LU (Ler UV-B treated); FC ( fah1 control); FU ( fah1 UV-B treated).
water and osmotic potentials and UV-B induction of stress-related proteins and compatible osmolytes. AC K N OW L E D GE M E N T S We thank B. M. Binges for technical assistance with the controlled environment chambers and UV-B exposure system, G. Parry (Dept. of Soil Science, University of Saskatchewan) for performing the stable carbon isotope analyses, and the Natural Sciences and Engineering Research Council of Canada for ®nancial assistance. L I T E R AT U R E C I T E D Aurora R, Wisniewski ME. 1994. Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica L. Batsch). II. A 60-kilodalton bark protein in cold-acclimated tissues of peach is heat stable and related to the dehydrin family of proteins. Plant Physiology 105: 95±101. Balakumar T, Hani Babu Vincent V, Paliwal K. 1993. On the interaction of UV-B radiation (280±315 nm) with water stress in crop plants. Physiologia Plantarum 87: 217±222. Bharti AK, Khurana JP. 1997. Mutants of Arabidopsis as tools to understand the regulation of phenylpropanoid pathway and UVB protection mechanisms. Photochemistry and Photobiology 65: 765±776. Caldwell MM, Flint SD. 1997. The use of biological spectral weighting functions and the need of scaling for the ozone reduction problem. Plant Ecology 128: 66±76. Close TJ. 1996. Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiologia Plantarum 97: 795±803. Close TJ. 1997. Dehydrins: a commonality in the response of plants to dehydration and low temperature. Physiologia Plantarum 100: 291±296. Close TJ, Raymond DF, Moonan F. 1993. A view of plant dehydrins using antibodies speci®c to the carboxy terminal peptide. Plant Molecular Biology 23: 279±286.
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Farquhar GD, O'Leary MG, Berry JA. 1982. On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves. Australian Journal of Plant Physiology 9: 121±137. Jansen MAK, Gaba V, Greenberg BM. 1998. Higher plants and UV-B radiation: balancing damage, repair and acclimation. Trends in Plant Science 3: 131±135. Knight JD, Livingston NJ, Van Kessel C. 1994. Carbon isotope discrimination and water-use eciency of six crops grown under wet and dryland conditions. Plant Cell and Environment 17: 173±180. Landry LG, Chapple CCS, Last RL. 1995. Arabidopsis mutants lacking phenolic sunscreens exhibit enhanced ultraviolet-B injury and oxidative damage. Plant Physiology 109: 1159±1166. Livingston NJ, Guy RD, Sun ZJ, Ethier GJ. 1999. The eects of nitrogen stress on the stable carbon isotope composition and water use eciency of irrigated and dry land white spruce (Picea glauca (Moench) Voss.) seedlings. Plant Cell and Environment 22: 281±289. Manetas Y, Petropoulou Y, Stamatakis K, Nikolopoulos D, Levizou E, Psaras G, Karaboumlotis G. 1997. Bene®cial eects of enhanced UV-B radiation under ®eld conditions: Improvement of needle water-relations and survival capacity of Pinus pinea L. seedlings during the dry Mediterranean summer. Plant Ecology 128: 100±108. Nedunchezhian N, Annamalainathan K, Kulandaivelu G. 1992. Induction of heat shock-like proteins in Vigna sinensis seedlings growing under ultraviolet-B (280±320 nm) enhanced radiation. Physiologia Plantarum 85: 503±506. Ormrod DP, Landry LG, Conklin PL. 1995. Short-term UV-B radiation and ozone exposure eects on aromatic secondary metabolite accumulation and shoot growth of ¯avonoid-de®cient Arabidopsis mutants. Physiologia Plantarum 93: 602±610. Ormrod DP, Schmidt A, Livingston NJ. 1997. Eect of UV-B radiation on the shoot dry matter production and stable carbon isotope composition of two Arabidopsis thaliana genotypes. Physiologia Plantarum 101: 497±502. Petropoulou Y, Kyparissis A, Nikolopoulos D, Manetas Y. 1995. Enhanced UV-B radiation alleviates the adverse eects of summer drought in two Mediterranean pines under ®eld conditions. Physiologia Plantarum 94: 37±44. Sambrook J, Fetsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Press. Smirno N, Cumbes QJ. 1989. Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28: 1057±1060. Sullivan JH, Teramura AH. 1990. Field study of the interaction between solar ultraviolet-B radiation and drought on photosynthesis and growth in soybean. Plant Physiology 92: 141±146. Taylor CB. 1996. Proline and water de®cit: ups, downs, ins and outs. The Plant Cell 8: 1221±1223. Teramura AH, Sullivan JH, Lydon J. 1990. Eects of UV-B radiation on soybean yield and seed quality: a 6-year ®eld study. Physiologia Plantarum 80: 5±11. Tevini M, Iwanzik W, Teramura AH. 1983. Eects of UV-B radiation on plants during mild water stress. II. Eects on growth, protein and ¯avonoid content. Zeitschrift fuÈr P¯anzenphysiologie 110: 459±467. Wanek W, Richter A. 1997. Biosynthesis and accumulation of Dononitol in Vigna umbellata in response to drought stress. Physiologia Plantarum 101: 416±424.
S H O R T CO M M U N I CAT I O N
The Interaction of Ultraviolet-B Radiation and Water De®cit in Two Arabidopsis thaliana Genotypes A N N A - M A RY S C H M I D T {, D O U G L A S P. O R M RO D{* , N I G E L J. L I V I N G S TO N { and S A N TO S H M I S R A { {Department of Biochemistry and Microbiology, Centre for Forest Biology, University of Victoria, P.O. Box 3020, Victoria, B.C., Canada, V8W 3N5 and {Department of Biology, Centre for Forest Biology, University of Victoria, P.O. Box 3020, Victoria, B.C., Canada, V8W 3N5 Received: 18 May 1999 Returned for revision: 28 September 1999 Accepted: 8 December 1999 It has been demonstrated, in both herbaceous and woody species, that tissue hydration resulting from exposure to drought is less pronounced if plants are concurrently exposed to ultraviolet-B radiation (UV-B). An explanation for the mechanisms underlying this phenomenon has been elusive. Arabidopsis thaliana (L.) Heynh. genotypes, defective in speci®c defences against UV-B exposure, may permit more insightful study of drought-UV-B interactions than is possible with genetically uniform plants. Arabidopsis has a rosette stature and has predominantly abaxial stomata. Thus, it is dicult to investigate its stomatal behaviour and gas exchange using conventional techniques and instrumentation. In this study, the relative abundance of 13C and 12C in leaf tissue (d 13C) was used as a means of determining water use eciency (WUE) and the relative balance, at the site of carbon ®xation, between CO2 supply and demand. UV-B insensitive (Ler) and sensitive ( fah1) Arabidopsis genotypes were raised in a growth chamber and exposed to 6 kJ m ÿ2 d ÿ1 UV-B irradiation and subjected to drought. In both genotypes, leaf desiccation was less pronounced than that of control plants that were subjected to drought but not exposed to UV-B. The relatively low (more negative) leaf d 13C values (indicating low WUE), but high dry matter production of the UV-B exposed plants suggest that their higher leaf water content was not primarily due to stomatal closure. We propose that the mechanisms underlying the maintenance of higher leaf water content involved UV-B and water stress induced biosynthesis of # 2000 Annals of Botany Company stress proteins and compatible osmolytes. Key words: Arabidopsis thaliana, ultraviolet-B, water de®cit, stable carbon isotopes, d dehydration, dehydrin.
I N T RO D U C T I O N All components of the biosphere are exposed to ultravioletB radiation (UV-B) at intensities that vary with the solar angle and the thickness of the stratospheric ozone layer (Jansen, Gaba and Greenberg, 1998). Research on the adaptation of plants to UV-B has revealed responses that suggest that UV-B is involved in the induction of multistress defences. Evidence of interactions between UV-B exposure and water stress in plants has emerged in recent years, but the mechanisms involved have received little attention. In ®eld-grown soybean, decreases in productivity following UV-B exposure were moderated by soil water de®cits (Sullivan and Teramura, 1990). The soybean cultivar most yield-sensitive to UV-B when well watered was least sensitive to UV-B when subjected to water stress. In contrast, a UV-B tolerant cultivar was sensitive to UV-B when also subjected to water stress (Teramura, Sullivan and Lydon, 1990). The interaction of soil water de®cit and UV-B stresses in cowpeas resulted in bene®ts from the combined stresses in terms of greater growth and * For correspondence. Fax (250) 721-7120, e-mail [email protected]
0305-7364/00/040571+05 $35.00/00
13
C, stomatal opening, tissue
development compared with exposure to single stresses (Balakumar, Hani Babu Vincent and Paliwal, 1993). Increased exposure to UV-B alleviated drought stress in Mediterranean pines (Petropoulou et al., 1995; Manetas et al., 1997). However, exposure to both UV-B and water stress led to decreased growth in cucumber and radish but protein content was increased by the combined stresses (Tevini, Iwanzik and Teramura, 1983). These initial reports indicated that both genotypic dierences and assimilate utilization are involved in the interaction of UV-B and water stress. The objective of this study was to investigate the interactive eects of exposure to drought and UV-B radiation on the growth and water relations of Arabidopsis. Speci®cally, we sought to determine whether exposure to UV-B limits tissue dehydration when plants are also subjected to drought stress. We hypothesized that such a response might come about because UV-B exposure stimulates the production of compatible osmolytes and stress proteins, thus allowing plants to maintain tissue turgor without closing their stomata. We speculated that this response would be more pronounced in a UV-B sensitive genotype. # 2000 Annals of Botany Company
572
Schmidt et al.ÐUV-B-Water Stress Interactions in Arabidopsis
Arabidopsis thaliana mutants defective in the synthesis of protective UV-B absorbing compounds have been widely used to study injury and defences against UV-B (Bharti and Khurana, 1997). The limited epidermal attenuation of UV-B radiation in these mutants, relative to that of wildtype genotypes, may help elucidate the mechanisms involved in UV-B interactions with other stresses in the underlying mesophyll tissue (Ormrod, Landry and Conklin, 1995). Our study utilized the fah1 mutant which, in contrast to the wild-type ecotype Landsberg erecta, lacks the ability to synthesize UV-absorbing sinapate esters and is thus highly sensitive to UV-B exposure (Landry, Chappel and Last, 1995). Because conventional gas exchange measurements are dicult to carry out on small plants such as Arabidopsis, we used measurements of tissue d 13C as an alternative. The relative abundance of 12C to 13C (d 13C) provides an integrated measure of Ci (the internal CO2 concentration) over the period during which CO2 is assimilated by the plant (Farquhar et al., 1982). Values of Ci vary among plants because of variation in stomatal conductance to CO2 and variation in photosynthetic capacity (chloroplast demand for CO2). Assuming that the concentration and isotopic composition of ambient CO2 is stable over the period of interest, the isotopic composition of leaf tissue should, therefore, re¯ect the balance between CO2 supply (and water lost) and assimilation. Discrimination against 13C typically decreases in response to the progressive depletion of rooting medium water that leads to stomatal closure and decreased Ci (Knight, Livingston and Van Kessel, 1994; Livingston et al., 1999). This is re¯ected in higher d 13C values and greater water use eciency (WUE). M AT E R I A L S A N D M E T H O D S Two independent experiments were performed, each using all combinations of two genotypes, two UV-B dosages, and two watering regimes. In each experiment, each of the eight treatment combinations had two replicate 12-cm diameter pots of each genotype of Arabidopsis thaliana (L.) Heynh. Ler and fah1 seedlings were propagated in a controlled environment chamber at 258C under cool white ¯uorescent lamps with a continuous photosynthetic photon ¯ux density of 150 mmol m ÿ2 s ÿ1. Weighed samples of seeds (1 mg 50 seeds) were suspended in tubes containing 0.1% (w/v) agar in water to facilitate uniform distribution of approx. 50 seeds on the surface of a wetted ®ne-textured synthetic rooting medium. Seeded pots were covered with transparent ®lm for 7 d. Seedlings were grown for a further 6 d after the ®lm was removed and then exposed for 10 d to 6 h d ÿ1 of either 0 UV-B (Mylar ®lm between lamps and plants) or 6 kJ m ÿ2 s ÿ1 (cellulose acetate ®lm between lamps and plants) biologically eective UV-B (Caldwell and Flint, 1997) from UV-B-emitting ¯uorescent lamps (UV-B 313, Q-Panel, Cleveland, OH, USA) added to the cool white ¯uorescent lamps. Complete nutrient solution was added to all pots from 11 to 13 d after seeding. Two rooting medium watering regimes were administered. Half the pots were wetted to ®eld capacity every second day; water was withheld from the other pots 14 d
after seeding. This discontinuation of watering, calibrated for the pot size, rooting medium and plant numbers, resulted in the onset of measurable leaf dehydration in control plants by the sixth day (19 d after seeding) of the UV-B exposure period. Seedlings were thinned to 30 per pot just before dierential treatments were initiated. The shoots of ®ve randomly selected seedlings were removed at each of six harvests, starting at the end of the daily UV-B exposure 14 d from seeding ( pretreatment) and continuing every second day to 24 d from seeding (10 d of treatment). Fresh shoots were weighed immediately, dried at 708C for 48 h, and reweighed to determine shoot water content at harvest. Dried shoot samples from the ®rst experiment were pulverized and subsamples analysed for d 13C by continuous ¯ow isotope ratio mass spectroscopy on a TracerMass mass spectrometer interfaced with a RoboPrep sample preparation unit (Europa Scienti®c, Crewe, UK). A peptide with the sequence CTGEKKGIMDKIKEKLPGQH was synthesized at the University of Victoria Protein Micro-Chemistry Centre, Victoria, Canada. This synthetic peptide containing the dehydrin carboxy terminus consensus sequence KIKEKLPG (Close, Raymond and Moonan, 1993) was used to produce antibodies. The peptide was coupled to maleimide-activated KLH carrier protein using the Imject1 kit according to the manufacturer's directions. The peptide-KLH conjugate was mixed with Freunds adjuvant and injected into rabbits; antipeptide antibody titres and speci®city were determined by ELISA with the free dehydrin peptide. Protein extraction and immunoblot analysis utilized the same growing conditions as above. Shoots for protein analysis were harvested 7 d after the beginning of UV-B treatment and immediately frozen in liquid nitrogen and stored at ÿ808C. There were control and UV-B treated plants only, and no water stressed plants. Total heat-stable protein extracts were prepared by a modi®cation of the method of Close et al. (1993). Tissue samples stored at ÿ808C were ground to a ®ne powder in liquid nitrogen with a mortar and pestle. Extraction buer (30 mM TES, pH 7.5; 50 mM NaCl; 0.5 mM PMSF) was added to ground tissue in a microfuge tube, vortexed and centrifuged at 13 800 g for 20 min. The supernatant was incubated at 908C for 30 min, transferred to ice, then centrifuged at 9000 g for 10 min at 48C. Heat stable proteins contained within the supernatant were quanti®ed by Bio-Rad protein assay. Duplicate SDS-PAGE gels were prepared and run according to Sambrook, Fetsch and Maniatis (1989). One gel was transferred to a nitrocellulose membrane using a Bio-Rad MiniProtein II Wet Blotting system, blocked with 3% (w/v) skim milk in TBS buer and incubated with rabbit anti-dehydrin polyclonal antibodies. Membranes were then incubated with goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma). Secondary antibody was detected using 4-nitroblue-tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP). Duplicate gels were Coomassie stained. Immunoblot analysis using pre-immune serum was performed, and no proteins were detected.
Schmidt et al.ÐUV-B-Water Stress Interactions in Arabidopsis R E S U LT S A N D D I S C U S S I O N Well watered Arabidopsis plants had growth responses to UV-B as would be expected for these genotypes (Landry et al., 1995). The shoot dry weight of Ler was unaected by UV-B while that of fah1 was decreased by 25±30% (Table 1). The shoot water content at harvest was uniformly near 90% in all well-watered treatments. In contrast, in plants subjected to drought, there were signi®cant declines in both water content and dry matter and clear dierences between UV-B treatments. These became most pronounced after 8 d of drought treatment. Six days after drought was imposed, large dierences in water content were not seen between genotypes across the UV-B treatments, but, within a given genotype, those plants not exposed to UV-B had the lowest water contents (Table 1). Dierences in dry matter production of droughtstressed plants across UV-B treatments were not signi®cant within genotypes. After 8 d, the shoot water content of drought-stressed fah1 plants exposed to UV-B was almost six-times as high as those that were not exposed. Despite these dierences in water content, there was no signi®cant dierence in dry matter production. Shoot water contents of Ler plants were lower than those measured after 6 d, but there was not a signi®cant dierence in water content between UV-B treatments. The d 13C data indicate that UV-B exposure, water de®cits and genotype had individual and interactive eects on carbon isotope discrimination (Fig. 1). In both genotypes, UV-B exposure resulted in greater discrimination against 13C (i.e. more negative d 13C values) in both well watered and drought-stressed plants. Dierences in sensitivity to UV-B exposure between genotypes became more pronounced over time. The lower d 13C values in fah1 plants, when exposed to UV-B, were consistent with their lack of protective UV-B absorbing compounds, and their lower productivity than Ler plants under either well watered or water stressed conditions. In both genotypes, the d 13C values for plants not exposed to UV-B but subjected to drought probably re¯ected stomatal closure
573
and hence a reduction in Ci. In both genotypes, dierences in d 13C between well watered and water stressed plants were less pronounced when plants were exposed to UV-B. This suggests that UV-B exposure might have oset some of the eects of water stress or vice versa. The low d 13C values in droughted fah1 plants exposed to UV-B suggests that the Ci in these plants was relatively high. There are alternative explanations. UV-B exposure might not have induced stomatal closure and therefore the supply of CO2 to the site of carboxylation was maintained. In a previous study we obtained results that suggested that exposure to UV-B did not induce stomatal closure in well-watered Ler or fah1 Arabidopsis (Ormrod, Schmidt and Livingston, 1997). If this was the case, the data suggest that UV-B exposure must have been associated with the production of osmolytes which allowed these plants (in marked contrast to those not exposed to UV-B) to maintain high water contents even when subjected to severe drought (Table 1). An alternative explanation is that exposure to UV-B might have suciently damaged the plant's photosynthetic machinery, leading to decreased demand for CO2 and hence high Ci. This latter explanation is unlikely because UV-B exposure did not bring about a signi®cant reduction in dry matter production in droughted plants (Table 1). Immunoblot analysis revealed that a protein (approx. 60 kDa), recognized by antibodies directed against a synthetic peptide containing the dehydrin consensus sequence, was induced by UV-B in both Ler and fah1 genotypes of Arabidopsis; levels of the protein increased nominally in UV-B treated Ler plants compared to the controls, whereas the increase in UV-B treated fah1 plants was far more substantial (Fig. 2). The accumulation of dehydrins in response to UV-B radiation may occur more rapidly than would a response to slow dehydration by water stress, contributing to the less pronounced water stress-induced tissue dehydration by concurrent exposure to UV-B radiation. As well, it has been proposed that dehydrins may act synergistically with compatible solutes to stabilize macromolecules, thereby stabilizing the protoplasm (Close, 1996).
T A B L E 1. Shoot dry weight and water content at harvest in well watered or drought stressed A. thaliana genotypes Ler and fah1 subjected to 0 or 6 kJ m ÿ2 d ÿ1 UV-B irradiation Well watered
Drought stressed
UV-B dose (kJ m ÿ2 d ÿ1)
Dry weight (mg per plant)
Water content (%)
6d Ler Ler fah1 fah1
0 6 0 6
5.54 + 0.22a 5.37 + 0.20a 5.32 + 0.50a 3.94 + 0.31b
90.4 + 0.2a 90.3 + 0.5a 90.5 + 0.2a 90.2 + 0.2a
4.42 + 0.55ab 4.15 + 0.53abc 3.09 + 0.37c 3.62 + 0.14bc
85.3 + 0.5b 87.9 + 0.6a 83.9 + 0.4b 88.1 + 1.0a
8d Ler Ler fah1 fah1
0 6 0 6
6.97 + 0.22b 6.36 + 0.33b 8.40 + 0.41a 5.80 + 0.45b
89.9 + 0.3a 89.6 + 1.0a 89.8 + 0.3a 89.3 + 0.0a
3.60 + 0.50b 5.04 + 0.49a 3.23 + 0.30b 3.40 + 0.23b
55.8 + 16.7a 69.2 + 9.4a 12.7 + 3.1b 75.2 + 6.4a
Duration Genotype
Dry weight (mg per plant)
Water content (%)
Means of two experiments + s.e. Mean separation within durations and watering regimes by paired comparison t-tests (P 0.05). Means followed by the same letter within 6 or 8 d and dry weight or water content are not signi®cantly dierent.
574
Schmidt et al.ÐUV-B-Water Stress Interactions in Arabidopsis −UV
6 days −32
+W
−W
−UV
+UV +W
−W
−32
−34 −35 −36
A Ler
−36
−UV +W
−W
+W
−W
−32
+W
−W
+UV +W
−W
−33 δ13C (%0)
δ13C (%0)
B fah 1
−UV
+UV
−33 −34 −35 −36
−34 −35
8 days −32
−W
+W
−33 δ13C (%0)
δ13C (%0)
−33
−W
+W
+UV
−34 −35
C Ler
−36
D fah 1
FIG. 1. Stable carbon isotope composition (d 13C) of shoots after 6 and 8 d of treatment in control (ÿUV) and UV-B irradiated (UV) Arabidopsis thaliana genotypes (A, C) Ler and (B, D) fah1. Plants were either watered to ®eld capacity every second day (w) or subject to drought stress (ÿw). Means and s.e.s of two replicate pots, ®ve shoots per pot, from the ®rst experiment.
Both Ler and fah1 genotypes, when subjected to drought but exposed to UV-B, maintained relatively high shoot water contents (Table 1), even though the d 13C data indicate that there was not substantial stomatal closure. Thus it is likely that leaf water content was maintained as a result of the induction by UV-B of stress-related proteins and osmotic adjustment. The production and accumulation by plants of low-molecular-weight solutes, called compatible osmolytes, is a widespread adaptive response to water stress. These organic compounds are osmotically active and include sugar alcohols, some amino acids and quaternary ammonium compounds (Taylor, 1996). They have important roles in osmotic adjustment, protection of macromolecules and metabolite storage during times of stress (Wanek and Richter, 1997). Most compatible osmolytes are also eective hydroxyl radical scavengers (Smirno and Cumbes, 1989) and help protect cells against the hydroxyl radicals generated by both water and UV-B stress. The accumulation of compatible osmolytes allows cell turgidity to be maintained in the face of water de®cits in the rooting medium. Aside from compatible solute accumulation, biochemical adaptations to stress also include changes in gene expression. Emerging evidence indicates that numerous environmental cues (dehydration, heat shock, low temperatures)
cause similar proteins to accumulate. Examples of such stress-related proteins include members of the dehydrin protein family and various families of heat shock proteins. Dehydrins are among the more commonly identi®ed proteins induced by water de®cits. In addition, a relationship between dehydrin accumulation and adaptation to stresses that have a dehydrative component such as freezing and cold stress has been reported (Arora and Wisniewski, 1994; Close, 1996, 1997). Our ®nding in this and the previous study (Ormrod et al., 1997), that UV-B exposure does not appear to induce pronounced stomatal closure in Arabidopsis, implicates a potential dehydrative eect in UV-B response. The induction of proteins similar to heat shock proteins has been reported in Vigna sinensis seedlings exposed to UV-B (Nedunchezhian, Annamalainathan and Kulandaivelu, 1992). Large increases in the levels of three groups of proteins in the range of 70, 53 and 16 kDa, similar to those observed after heat shock treatment, were observed. UV-B radiation may provide a certain amount of cross-adaptation to other environmental stresses and, like other stresses, may induce sets of proteins having a protective role. Our results strongly suggest that UV-B exposure moderates the eects of water stress and demonstrate the need for further research to establish the relations between plant
Schmidt et al.ÐUV-B-Water Stress Interactions in Arabidopsis LC
LU
FC
FU
kDa 62 > 47.5 >
32.5 > FIG. 2. Immunoblot of dehydrin proteins present in Arabidopsis genotypes Ler and fah1; 5 mg total protein loaded per lane. LC (Ler control); LU (Ler UV-B treated); FC ( fah1 control); FU ( fah1 UV-B treated).
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